The thyrotropin (TSH) receptor (TSHR) is the primary antigen of Graves'disease and the target for thyroid stimulating 'TSHR antibodies (TSHR-Ab)found in the serum of such patients. TSHR-Ab are characterized by their prolonged action and were originally called Long Acting Thyroid Stimulators (LATS). In this application, we seek to continue our successful cloning and characterization of monoclonal antibodies to the TSH receptor which have resulted in the first monoclonal antibody with stimulating activity. In particular, monoclonal antibody MS-1 has shown potent TSHR stimulating activity when tested with CHO cells stably expressing hTSHR with activity evident down to 2ng/ml ofIgG. The specific aims of this competitive renewal are: Aim 1: Identify additional monoclonal thyroid-stimulating antibodies. We will first continue to build on our success by developing an expanded panel of monoclonal antibodies to the TSH receptor with thyroid stimulating activity. Aim 2: Define TSHR-AB epitopes. We will characterize the linear and non-linear antigenic epitopes for the derived TSH receptor antibodies to define the major immunogenic 'regions (MIRs) on the TSH receptor. Aim 3: Defining antibody-mediated regulation of TSHR post-translational modifications. We will co-express TSHRcpp TSHRmycconstructs in CHO cells and examine whether occupation of the receptor molecule by TSHR-Abs will modulate the FRET index as we have seen for TSH ligand. In addition, we have recently developed a unique assay for measuring TSHR cleavage and will examine the influence of TSHR-Abs on receptor cleavage with the aim of gaining insight into the functional role of such cleavage in thyroid autoimmunity. Aim 4: Examine the biologic function and signal transduction induced by TSHR-Abs. We will examine the specificity of TSHR-Abs with regard to their growth stimulating/inhibiting potential and their activarion/inacrivation of differentiated thyroid cell function. The isolation and characterization of antibodies to the TSH with similar characteristics to the TSHR-Abs found in the serum of Graves'disease patients will provide major new insight into their mechanisms of action and offer novel approaches to the control of thyroid dysfunction.